o:37319 Application of VHH-Immobilized Cryogel-Based Immunoaffinity Chromatography for Isolation of Extracellular Vesicles en Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow cytometry. Proteomic profiling of the isolated samples identified 234 proteins, of which 63% were ExoCarta-listed exosomal proteins; contaminants such as albumin and apolipoproteins were also identified. The purification method provided samples with ~2 × 109 EVs/mL, with EV median size of 135 nm and consistent protein-to-lipid ratio across three independent isolations (CV < 10%). This study demonstrates that VHHfunctionalized cryogels (VHH-SMC) are a rapid and reproducible EV purification method that represents a promising alternative to conventional ultracentrifugation- or precipitationbased protocols. While optimization of nanobody density and reduction in plasma protein carryover are still necessary, the platform holds potential for scalable EV enrichment, a condition that can significantly speed up biomarker research and clinical diagnostics. extracellular vesicles; cryogels; nanobodies; immunoaffinity chromatography 1552099 https://doi.org/10.3390/ molecules30224337 2025-12-23T13:04:20.597Z 44 yes 46 Jovana Terzić Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, University of Belgrade, 11000 Belgrade Lidija Filipović Innovative Centre of the Faculty of Chemistry, 11158 Belgrade, Serbia person Ninoslav Mitić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor bioloških nauka person 0000-0003-2756-6517 Sanja Stevanović Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, University of Belgrade, 11000 Belgrade person Jugoslav Krstić Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, University of Belgrade, 11000 Belgrade person Ario de Marco Laboratory for Environmental and Life Sciences, University of Nova Gorica, 5000 Nova Gorica, Slovenia person Julie Courraud Proteomics Core Facility, School of Science, National and Kapodistrian University of Athens, 15784 Athens, Greece; Section of Clinical Therapeutics, Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, 15771 Athens, Greece person Milica Popović Faculty of Chemistry, University of Belgrade, 11158 Belgrade, Serbia person application/pdf 3391639 https://phaidrabg.bg.ac.rs/o:37319 no yes 16 70 92000001 11A24 11A19 11A29 11A2903 Application of VHH-Immobilized Cryogel-Based Immunoaffinity Chromatography for Isolation of Extracellular Vesicles 4337 30 Molecules Basel, Switzerland MDPI 2025